Antigen discovery has focused on using human serum samples collected from individuals that are naturally exposed to malaria and appear to develop effective immunity that prevents gametocytemia or blocks parasite transmission to mosquitoes, as tools to identify candidate vaccine antigens. Specifically, serum samples that have activity of interest are compared to sera that lack this activity, for their ability to select or recognize individual recombinant proteins constructs in P. falciparum expression libraries. Recombinant proteins identified through differential screening are then prepared as immunogens and tested for their ability to induce effective anti-gametocyte or transmission-blocking antibodies. Highlighted in this years summary are results from our publications: Antibodies to PfsEGXP, An Early Gametocyte Enriched Phosphoprotein, Predict Decreased Plasmodium falciparum Gametocyte Density In Humans. To identify novel anti-gametocyte TBV antigens, we partnered with our collaborators at Brown University to interrogate the gametocyte proteome with their whole proteome differential screening method using plasma from a treatment-reinfection study that LMIV scientists had conducted in western Kenya previously. At the start of the high transmission season, 144 males (12-35 years) were enrolled in that study, treated with quinine and doxycycline, peripheral venous blood sample were obtained, and volunteers were followed with weekly blood films for 18 weeks to quantify gametocytemia. Using plasma pooled from individuals with low versus high gametocyte carriage, we differentially screened a P. falciparum gametocyte stage cDNA expression library. We identified eight parasite genes uniquely recognized by gametocyte-resistant but not by gametocyte-susceptible individuals. Antibodies to one of these antigens, PfsEGXP, predicted lower gametocytemia measured over the 18-week transmission season (P = 0.021). When analyzed dichotomously, anti-PfsEGXP responders had 31% lower gametocyte density over 18 weeks of follow-up, compared to non-responders (P = 0.04). PfsEGXP is one of the first reported gametocyte-specific target of antibodies that predict decreased gametocyte density in humans and supports our novel TBV antigen discovery platform. Safety and immunogenicity of Pfs25H-EPA/Alhydrogel, a transmission blocking vaccine against Plasmodium falciparum: a randomized, double-blind, comparator-controlled, dose-escalation study in healthy malaria exposed Malian adults. Pfs25H-EPA is a protein-protein conjugate transmission-blocking vaccine against Plasmodium falciparum that is safe and induces functional antibodies in malaria-naive individuals. In this field trial, we assessed Pfs25H-EPA/Alhydrogel for safety and functional immunogenicity in Malian adults. The primary outcomes were safety and tolerability for all vaccinees. The secondary outcome measure was immunogenicity 14 days after vaccination in the per-protocol population, as confirmed by the presence of antibodies against Pfs25H measured by ELISA IgG and antibody functionality assessed by standard membrane feeding assays and by direct skin feeding assays. Pfs25H-EPA/Alhydrogel was well tolerated and induced significant serum activity by standard membrane feeding assays but transmission blocking activity was not confirmed by weekly direct skin feed. This activity required four doses, and titres decreased rapidly after the fourth dose. Alternative antigens or combinations should be assessed to improve activity, and emphasizes the importance of our efforts at TBV antigen discovery. In addition to these published findings, we undertook novel approaches to identify new TBV antigens during the past fiscal year. In FY2018, a process was developed to recover IgG light and heavy chain genes from human B-cells and express full-length human monoclonal antibodies derived from all germline lineages. Tetramers generated from biotinylated Pfs230 were used to pull down B-cells from samples of 5 patients immunized with Pfs230 in Alhydrogel. The B cells were sequenced and 2 recombinant antibodies were generated. Both antibodies were reactive to Pfs230D1 as measured by ELISA. Additionally, one of the antibodies was shown to block parasite transmission in a complement-dependent manner in the standard membrane feeding assay. Additionally, mice were immunized with Pvs230D1 (from P. vivax) for production of recombinant antibodies against Pvs230D1. Also in FY2018, full-length Pfs230 constructs were synthesized, cloned and transfected into a mammalian expression system for production of full-length Pfs230. Full length antigen will facilitate efforts to identify new Pfs230-based immunogens that could be assessed as TBV.